The transparent cornea is made up of an anterior stratified epithelium, a collagenous stromal matrix containing fibroblasts called keratocytes, Descemet's membrane and a single-layered endothelium. We have put forth the refracton hypothesis attempting to relate cornea to lens. The cornerstone of this hypothesis is that the exceptionally abundant intracellular proteins in the cornea share properties with the multifunctional crystallins of the lens. Our current corneal research addresses the molecular basis for corneal-specific gene expression, the roles of the abundant, intracellular corneal proteins, the global patterns of corneal gene expression, and stratification of the corneal epithelium. The advances in FY2004 include the following. Mouse aldehyde dehydrogenase 3 (ALDH3a1) comprises half of the water-soluble protein in the corneal epithelial cells. This year we found that the -2537/+3486 promoter fragment (containing exon 1, intron 1 and part of exon 2) of the ALDH3a1 gene is activated in co-transfection tests in COS7 cells by Pax6, KLF4, KLF5, Junb and POU2f1/Oct-1 (all enriched in the corneal SAGE library produced last year). Some of these activating transcription factors are also used for crystallin gene expression in the lens, consistent with the refracton hypothesis linking lens and cornea. Pax6 stimulation is blocked by interferon regulatory factor 1. Putative Pax6 binding sites within the promoter and intron 1 are being studied further by mutagenesis and gel shift assays. A ~400 bp negative regulatory element was found in intron 1 which represses a heterologous promoter in a position and orientation-dependent fashion. An explanted rat corneal transfection system was developed to facilitate study of the regulation of corneal gene promoters in a bonafide corneal background. In collaboration with Dr. Jerome Kuszak (Rush Presbyterian Hospital, Chicago), we found significant reductions in microplicae in the corneal surface epithelial cells of the ADLH3a1 null mice, indicative of increased cell sloughing. Small spaces between corneal epithelial cells (not present in wild type mice) were apparent, suggestive of adhesive defects. Microarray data indicated that ~50% of the differentially regulated genes in the ALDH3a1 null corneas are those controlled by interferon. Together, these data suggest that barrier function of the corneal epithelium is compromised causing an immune response. A sideline of translational interest is that the ALDH3a1 null mice contain Helicobacter (which can lead to ulcers and cancer), while wild type mice do not. Preliminary experiments in collaboration with Dr. Vasilis Vasiliou (University of Colorado Health Sciences Center, Denver) using the ALDH3a1 null mice show elevated protein conjugates of 4-hydroxy-nonenal, a substrate of ALDH3a1, consistent with a detoxifying function for this enzyme. We previously reported that gelsolin comprises half of the water-soluble protein of the zebrafish cornea, and that a different gelsolin is expressed ubiquitously in the zebrafish at lower levels. In FY2004 in collaboration with Dr. Eugene Koonin?s laboratory (NLM, NIH) we established that the corneal-preferred gelsolin-like protein in zebrafish clusters with the adseverins while the ubiquitously expressed gelsolin clusters with the authentic gelsolins. The corneal-preferred adseverin is also expressed in early embryogenesis and contributes to dorsal-ventral signaling. By contrast, expression of the gelsolin gene was preferentially associated with vascular development and is not associated with dorsal-ventral signaling. Preliminary results suggest that a 2.5 Kb promoter fragment of the adseverin gene drives the expression of a reporter gene in the embryonic eye of microinjected zebrafish eggs. Inclusion of exon 1, intron 1 and part of exon 2 in a 4 Kb promoter fragment augments eye expression but does not eliminate it in surrounding tissues. Further constructs are being made including upstream and downstream sequences. In FY2004 we constructed a newborn mouse corneal SAGE library to compare with postnatal 9 and 6-week-old libraries which were made last year. 300 transcription factors (many differentially regulated during postnatal corneal development) were represented in the 3 libraries. Expression patterns and developmental functions for 4 corneal-enriched factors [Pax6, Kruppel-like factor 4 (KLF4), interferon regulatory factor-1 (IRF1) and ELF3] are being determined during development by in situ hybridization and by microinjection of morpholino oligonucleotides (MOs) into fertilized zebrafish eggs. Preliminary results showed a 12% decrease in eye size and 5% loss of eyes in embryos co-injected with MOs to Pax6a and Pax6b (zebrafish have 2 Pax6 genes). FY2004 also showed progress in the analysis of earlier SAGE data on global gene expression in the limbal stem cells and central epithelial cells of the adult rat. In collaboration with Dr. Michael Bustin (NCI, NIH), we showed that HMGN1 (a multifunctional high mobility group protein) null mice have reduced corneal epithelial cells, altered differentiation reflected by changes in epithelial cell layer-specific markers, and adhesive defects in the superficial corneal epithelial cells. Transketolase, cytokeratin 12 and ALDH3a1 gene expression were not altered. p63, a transcription factor considered a master regulator of epidermal differentiation, co-localized with HMGN1 in clumps of basal epithelial cells across the entire cornea in wild type mice at PN25; p63 was confined to peripheral corneal epithelial cells in PN25 HMGN1 null mice, similar to the pattern observed in aging wild type corneas. Finally, KLF4 has been deleted specifically in the cornea of mice using floxed genes (KLF4-loxP mice obtained from Dr. Klaus Kaestner, University of Pennsylvania School of Medicine) and Le-Cre mouse (obtained from Dr. Peter Gruss, Max Planck Institute, Germany). In preliminary experiments corneal deletion of KLF4 produced a small eye phenotype with defects in lens, cornea and iris. The goals of this study are to define the role of KLF4 on corneal development and to identify their corneal target genes.